The substrate specificity, kinetic properties, and catalytic mechanism of folylpoly-gamma-glutamate synthetases are under investigation. The enzyme has been purified to homogeneity from Corynebacterium, Lactobacillus, Escherichia coli, and hog liver. The E. coli synthetase gene has been isolated and cloned into high-expression vectors. The sequence of the gene and its regulatory region are under study. Various pteroylmono-\and polyglutamate analogs have been synthesized and are being used to map the folate binding sites of these enzymes. Inhibitors of the enzymes have been synthesized, and additional potential inhibitors are in preparation. Inhibitors of folylpolyglutamate synthetase are being tested for their ability to cause a cellular folate depletion by preventing the formation of pteroylpolyglutamates, the forms of the vitamin that are selectively retained by tissues. The ability of these inhibitors to increase the exogenous requirement for products of one carbon metabolism and to potentiate any differences in nutritional requirements between tumorigenic and nontumorigenic cells are under study. The enzymatic conversion of antifolates to polyglutamate forms and the effects of this metabolism on the cytotoxic action of these drugs will be investigated. (B)